Many rapid lateral flow diagnostic tests are in use throughout the world today. Rosenstein was the first to describe the methodology (U.S. Pat. No. 5,591,645) in 1987, and many others have contributed improvements since then (U.S. Pat. No. 4,855,240, Rosenstein et al.; U.S. Pat. No. 5,602,040, May et al.; U.S. Pat. No. 5,714,389, Charlton et al.; U.S. Pat. No. 5,824,268, Bernstein et al.; and U.S. Pat. No. 7,189,522, Esfandiari). However, none of these tests that detect antibodies to specific antigens provide a means within each test to simultaneously detect whether the sample being evaluated actually contains a sufficient amount of immunoglobulin to allow detection of specific antibodies, if present. Also, none of these tests currently available have built-in controls to simultaneously report immunoreactivity of the critical reagents required for the test to provide accurate results. These two important omissions mean that some tests may provide falsely negative results due to inadequate sample or failed reagents. When a person has HIV antibodies, and they provide an inadequate sample to the rapid test for evaluation, or when the critical reagents of the test have deteriorated, no lines will develop that would normally indicate a positive result, such as antibodies in the test sample to HIV antigen on the test membranes. The test user is likely to incorrectly conclude that the test is negative because no lines have appeared. However, had controls for sample adequacy and reagent reactivity been included in the test, this mistake would not occur. The user would observe that either the sample was inadequate for testing, or that critical reagents required for accurate test performance were not working, and make the correct conclusion that the test result was INVALID and could not be interpreted, rather than falsely concluding that the test was negative.
Specific examples of this type of failure of rapid tests for HIV have been reported in Kenya and Uganda (Mar. 28, 2009—Uganda Monitor Online, Dr. Z. Akol, the STD/AIDS Control Programme manager at the Health Ministry, Kampala, Uganda) and noted failures of tests given to 6,255 people due to exposure to harsh storage conditions and using tests beyond their expiration dates, failures that would be detected with controls for antigen and critical reagent reactivity, but not without, and also failures due to errors the tests short reading windows of 20 minutes or less, reading beyond which may give false positives. In India, more than one hundred thousand persons were tested in five states with faulty tests that lacked controls to detect reagent failure or sample inadequacy—76,464 persons were tested in Mumbai alone, in the period 2-07 through 5-07, and S Kudalkar, chief of the Mumbai District AIDS Control Society (MDACS) stated “It is those who tested negative in those three months who are a cause for worry. Since they aren't tested again, if their result is wrong, they could have missed appropriate treatment.” Dr. A. Deshpande also pointed out that pregnant mothers with HIV infection may have been missed. The Delhi High Court ruled on May 25, 2009 that all those tested between February-07 and May-07 must be retested due to the poor quality of the HIV rapid tests used (M. Rajadhyaksha, TNN 29 Jul. 2009).
Many current rapid lateral flow tests contain a so-called positive control, which is nothing more than a incomplete procedural control that simply reports that fluid introduced into the rapid test has migrated to the end of the test results window (see U.S. Pat. No. 5,989,921, Charlton et al., claim 1, line 26, “said control site comprising an immobilized binder that binds said conjugate,” regardless of whether antibody is present or absent in the sample. Also see U.S. Pat. No. 5,656,503, May et al., claim 42 (d), line 63 “a control zone downstream from said test result zone in said dry porous carrier for binding labeled reagent to indicate that said applied liquid biological sample has been conveyed by capillarity beyond said test result zone.” Again, the binding is independent of the presence or absence of the analyte of interest in the liquid biological sample. Also see OraSureOraquick ADVANCE Rapid HIV-1/2 test PMA, that refers to “a goat anti-human IgG procedural control immobilized onto a nitrocellulose membrane in the . . . . Control (C) zone” . . . “This built-in procedural control serves to demonstrate that a specimen was added to the vial and that the fluid has migrated adequately through the test device”. These controls do not catch false negative results that may result from testing an inadequate sample or failure of critical test reagents.
Current advanced instrument based tests for detection of antibodies to HIV typically will detect the presence of antibodies to HIV a few days sooner during seroconversion following infection than can be detected with rapid lateral flow diagnostic tests, and well before Western Blot confirmatory tests become positive. It would be desirable for rapid lateral flow diagnostic tests to have increased sensitivity that allowed them to more closely approximate the sensitivity of instrument based tests.
Current rapid lateral flow diagnostic tests for HIV are approved by the US FDA for use with only very short reading windows, beyond which the test must not be interpreted due to an increased risk of false positive results. For example, the Trinity Biotech UniGold Rapid HIV-1 antibody test must be read over a 2 minute window, precisely 10-12 minutes after test initiation. The Inverness Determine HIV-1/2 antibody test must be read over a 5 minute window between 15 and 20 minutes, and the OraSureOraQuick ADVANCE HIV-1/2 test must be read over a 20 minute window between 20 and 40 minutes. In busy emergency room or urgent care clinics, this limited reading period for the rapid tests can be difficult to achieve, resulting in either failure to read the test appropriately, or failure of widespread use of the test due to its limitations. It would be desirable to have a test with a reading window of at least several hours, and preferably several days to months.
Currently existent rapid lateral flow diagnostic test devices occasionally permit backflow of test reagents from the downstream absorbent pad, upstream into the reading window. When this occurs, the test results are obscured and accurate reading of the results becomes impossible. It would be desirable to have a method and device that utilized the method that prevents such backflow of reagents.